The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Researchers have been developing methods for cloning mammalian animals over the past two decades. These reported methods typically include the steps of (1) isolating a cell, most often an embryonic cell; (2) inserting the cell or nucleus isolated from the cell into an enucleated oocyte (e.g., the oocyte's nucleus was previously extracted), and (3) allowing the embryo to mature in vivo.
The first successful nuclear transfer experiment using mammalian cells was reported in 1983, where the pronuclei isolated from a murine (mouse) zygote were inserted into an enucleated oocyte and resulted in like offspring(s). McGrath & Solter, 1983, Science 220:1300-1302. Subsequently, others described the production of chimeric murine embryos (e.g., embryos that contain a subset of cells having significantly different nuclear DNA from other cells in the embryo) using murine primordial germ cells (PGC). These cells are and can give rise to pluripotent cells (e.g., cells that can differentiate into other types of cells but do not differentiate into a grown animal). Matsui et al., 1992, Cell 70:841-847 and Resnick et al., 1992, Nature 359:550; Kato et al., 1994, Journal of Reproduction and Fertility Abstract Series, Society For the Study of Fertility, Annual Conference, Southampton, 13:38.
Some publications related to murine pluripotent cells stress the importance of steel factor for converting precursor cells into pluripotent cells. U.S. Pat. Nos. 5,453,357 and 5,670,372, entitled "Pluripotent Embryonic Stem Cells and Methods of Making Same," issued to Hogan. These same publications indicate that murine pluripotent cells exhibit strong, uniform alkaline phosphatase staining.
Although murine animals were never clearly cloned from nuclear transfer techniques using embryonic cells, some progress was reported in the field of cloning ovine (sheep) animals. One of the first successful nuclear transfer experiments utilizing ovine embryonic cells as nuclear donors was reported in 1986. Willadsen, 1986, Nature 320:63-65. A decade later, others reported that additional lambs were cloned from ovine embryonic cells. Campbell et al., 1996, Nature 380:64-66 and PCT Publication WO 95/20042. Recently, another lamb was reported to be cloned from ovine somatic mammary tissue. Wilmut et al., 1997, Nature 385:810-813. Some methods for cloning ovine animals focused upon utilizing serum deprived somatic ovine cells and cells isolated from ovine embryonic discs as nuclear donors. PCT Publications WO 96/07732 and WO 97/07669. Other methods for cloning ovine animals involved manipulating the activation state of an in vivo matured oocyte after nuclear transfer. PCT Publication WO 97/07668.
While few lambs were produced, publications that disclose cloned lambs report a cloning efficiency that is, at best, approximately 0.4%. Cloning efficiency, as calculated for the previous estimate, is a ratio equal to the number of cloned lambs divided by the number of nuclear transfers used to produce that number of cloned lambs.
Despite the slower progress endemic to the field of cloning bovine animals, a bovine animal was cloned using embryonic cells derived from 2-64 cell embryos. This bovine animal was cloned by utilizing the nuclear transfer techniques set forth in U.S. Pat. Nos. 4,994,384 and 5,057,420. Others reported that cloned bovine embryos were formed by nuclear transfer techniques utilizing the inner cell mass cells of a blastocyst stage embryo. Sims & First, 1993, Theriogenology 39:313 and Keefer et al., 1994, Mol. Reprod. Dev. 38:264-268. In addition, another publication reported that cloned bovine embryos were prepared by nuclear transfer techniques that utilized PGCs isolated from fetal tissue. Delhaise et al., 1995, Reprod. Fert. Develop. 7:1217-1219; Lavoir 1994, J. Reprod. Dev. 37:413-424; and PCT application WO 95/10599 entitled "Embryonic Stem Cell-Like Cells." However, the reports demonstrated that cloned PGC-derived bovine embryos never clearly developed past the first trimester during gestation. Similarly, embryonic stem cell (e.g., cell line derived from embryos which are undifferentiated, pluripotent, and can establish a permanent cell line which exhibits a stable karyotype), ESC, derived bovine embryos never developed past fifty-five days, presumably due to incomplete placental development. Stice et al., 1996, Biol. Reprod. 54: 100-110.
Despite the progress of cloning ovine and bovine animals, there remains a great need in the art for methods and materials that increase cloning efficiency. In addition there remains a great need in the art to expand the variety of cells that can be utilized as nuclear donors, especially expanding nuclear donors to non-embryonic cells. Furthermore, there remains a long felt need in the art for karyotypically stable permanent cell lines that can be used for genome manipulation and production of transgenic cloned animals.